Long-range single-molecule mapping of chromatin accessibility in eukaryotes

Abstract

Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N6-methyladenosine (m6A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.

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Fig. 1: The SMAC-seq assay for profiling chromatin accessibility and nucleosome positioning at the multikilobase scale.
Fig. 2: SMAC-seq provides a single-molecule linked-read view of the chromatin landscape.
Fig. 3: SMAC-seq’s single-molecule readout provides insights into the distribution and relationship between mutually exclusive chromatin yeast rDNA states.
Fig. 4: SMAC-seq provides a high-resolution strand-specific view of genomic occupancy by DNA-binding proteins and complexes.
Fig. 5: Coordinated changes in chromatin accessibility and nucleosomal occupancy during the yeast stress response.

Data availability

Short-read datasets associated with this study are available through GEO accession GSE128290. Nanopore data are available through SRA BioProject PRJNA594057. Nanopore raw data are available at https://zoharshiponh.s3.amazonaws.com/NMETH_2020/index.html.

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Acknowledgements

This work was supported by National Institutes of Health grants (nos. P50HG007735, RO1 HG008140, U19AI057266 and UM1HG009442 to W.J.G., 1UM1HG009436 to W.J.G. and A.K., 1DP2OD022870-01 and 1U01HG009431 to A.K.), the Rita Allen Foundation (to W.J.G.), the Baxter Foundation Faculty Scholar Grant and the Human Frontiers Science Program grant RGY006S (to W.J.G). W.J.G is a Chan Zuckerberg Biohub investigator and acknowledges grant nos. 2017-174468 and 2018-182817 from the Chan Zuckerberg Initiative. Z.S. is supported by EMBO Long-Term Fellowship EMBO ALTF 1119-2016 and by Human Frontier Science Program Long-Term Fellowship HFSP LT 000835/2017-L. G.K.M. is supported by the Stanford School of Medicine Dean’s Fellowship. N.A.S.A. is funded by the Department of Defense through a National Defense Science and Engineering Grant and by a Stanford Graduate Fellowship. We also thank members of the Greenleaf and Kundaje laboratories for their helpful suggestions and discussions on the subject over the course of the study.

Author information

Z.S., G.K.M. and N.A.S.A. conceived and designed the study. Z.S., G.K.M. and N.A.S.A. performed initial experiments. Z.S., M.P.S. and G.K.M. performed diamide time course experiments. G.K.M. and Z.S. analyzed data. W.J.G., A.K. and J.M.S. supervised the study. G.K.M., Z.S., W.J.G. and A.K. wrote the manuscript with input from all authors.

Correspondence to William J. Greenleaf.

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Peer review information Nicole Rusk and Lei Tang were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Shipony, Z., Marinov, G.K., Swaffer, M.P. et al. Long-range single-molecule mapping of chromatin accessibility in eukaryotes. Nat Methods (2020). https://doi.org/10.1038/s41592-019-0730-2

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